Clostridium difficile is an obligate anaerobic, spore-forming, gram positive bacterium that is a notorious pathogen for humans and other mammals (Bartlett et al., 1977; Bartlett et al., 1979; Keel et al., 2007; Sunenshine & McDonald, 2006). At low densities C. difficile can reside innocuously in the mammalian gastrointestinal (GI) tract, but upon expansion, frequently as the result of administered antibiotics reducing the commensal bacteria, C. difficile bacteria produce sufficient exotoxins to cause a range of diseases from a mild diarrheal disease to a characteristic pseudo-membranous colitis, which is life-threatening, particularly to older humans and others with significant co-morbidities (Bartlett, 2002).
Because spores formed by this pathogen disseminate widely and are difficult to eradicate or inactivate in hospitals and chronic care facilities, the probability of patients being colonized by C. difficile increases sharply upon their entering such a facility (Bartlett, 2007). In fact, a relatively new strain of C. difficile that is a hypervirulent toxigenic bacterial strain of C. difficile, BI/NAP1/027, which causes severe disease in massive outbreak settings, has recently been well documented (Spigaglia et al., 2002; Pépin et al., 2004; McDonald et al., 2005; Muto et al., 2005; Loo et al., 2005; Belmares et al., 2009). The incidence of C. difficile associated disease (CDAD) in children, previously at low risk, has also increased substantially (Benson et al., 2007; Zilberberg et al., 2010).
Eliminating the pathogen prophylactically in asymptomatic carrier or colonized subjects by administering antibiotics is strongly contraindicated because of the high risk of inducing C. difficile associated disease.
R-type bacteriocins made by gram negative bacteria have been described and have been deployed by such bacteria to kill other competitive gram negative strains, even in some circumstances other species or genera of gram negative bacteria (Kageyama et al., 1964; Kageyama et al., 1964a; Kingsbury, D, 1966; Blackwell and Law, 1981; Blackwell et al., 1982; Campagnari et al., 1994; Strauch et al., 2001; Jabrane et al., 2002). The fusion of base plate attachment regions (BPAR) of R-type pyocins to heterologous receptor binding domains (RBD), resulting in the creation of novel R-type pyocins with novel bactericidal specificities for gram negative bacteria has been described (Williams et al., 2008; Scholl et al., 2009).
Other high-molecular-weight bacteriocins or R-type bacteriocins have been described in gram-positive bacteria (Coetzee et al., 1968; Thompson and Pattee, 1981; Zink et al., 1995). However, much less is known about R-type high molecular weight bacteriocin structures produced by gram positive bacteria. But while such have been described, none has been characterized at a genetic level or manipulated in a manner supportive or necessary for developing a useful agent. High molecular weight bacteriocins have been described for 2 Clostridium species, botulinum and perfringens (Ellison and Kautter, 1970; Anastasio et al., 1971; Nieves et al., 1981). None has been described that is produced by C. difficile or kills C. difficile. 